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. 2021 Nov 23;13(3):759–788. doi: 10.1016/j.jcmgh.2021.11.007

Figure 2.

Figure 2

CRISPR/Cas9-mediated TM6SF2 ablation in HepG2 cells. (A) Schematic representation of the TM6SF2 sequence (gene ID: 53345; referred to as transcript variant 1: NM_001001524.3) highlighted the same clonal Cas9-induced indel mutations in both TM6SF2-/- and MBOAT7-/-TM6SF2-/- clones (blue) of 202 nucleotides (Δ202). The Cas9 cutting site is indicated by the symbol “|” (red), and the transcription start site (ATG) of the protein coding sequence (NP_001001524.2) is shown in green. (B) mRNA and protein expression of TM6SF2 was evaluated through reverse-transcription quantitative PCR and Western blot, respectively. TM6SF2 reduction was detected in TM6SF2-/- and MBOAT7-/-TM6SF2-/- cells. (C) MBOAT7 mRNA and protein levels were lower in MBOAT7-/- and MBOAT7-/-TM6SF2-/- cells compared with Cas9+ and TM6SF2-/- cells. (D) ApoB protein was assessed in cell supernatants by Western blot and normalized to the entire lane of the Ponceau stain. Either TM6SF2-/- or MBOAT7-/-TM6SF2-/- showed low ApoB levels. (E) TAG-rich lipoprotein secretion was measured in cell supernatants and normalized to levels of total cholesterol by using the Cholesterol Colorimetric Assay Kit–HDL and LDL/VLDL (Abcam). Both TM6SF2-/- and MBOAT7-/-TM6SF2-/- dampened TAG-rich lipoprotein release. Data were normalized to the β-actin housekeeping gene for reverse-transcription quantitative PCR and Western blot and they are expressed as means and SE. At least 3 independent experiments were conducted. Adjusted ∗P < .05 and ∗∗P < .01 vs Cas9+ and/or vs MBOAT7-/-. WT, wild-type.